Rice Glutelin Research Articles

Designing ferulic acid-functionalized dual protein conjugates: Process-induced changes, structural interplays, and sustainable calcium fortification

This study investigates the structural changes and covalent interactions of rice glutelin and egg white proteins with ferulic acid, considering the effects of germination and whisking times. Using ternary phase diagrams, we analyzed the antioxidant properties and in vitro digestive behaviors of these protein-ferulic acid conjugates. Increased germination time enhanced the conjugate potential of rice glutelin, while prolonged whisking time increased the molecular flexibility of egg white proteins. The optimal ratio of rice glutelin to egg white proteins (6:4) resulted in higher surface hydrophobicity. In vitro digestion showed that a higher proportion of rice glutelin aids gastric digestion, while more egg white protein supports intestinal hydrolysis. Extended germination time reduced particle size and antioxidant capacities during digestion but negatively impacted calcium loading from eggshells. These findings highlight the potential of tailored protein-ferulic acid conjugates for improved functional properties and food applications.

Characterization of ulvan polysaccharide extracted from Ulva pertusa and its effect on thermal, rheological, and gelling properties of rice flour

Three ulvan fractions (UPs 1–3) were extracted from Ulva pertusa via hot-water extraction. UP1 exhibited a molecular weight of 729,151 Da, while UPs 2 and 3 ranged from 19,952 to 750,384 Da. These fractions differed in monosaccharide, uronic acid, and sulfate levels. Zeta potentials for polysaccharide solutions (0.2–0.6 % w/v) ranged from −34.4 to −25.1, all demonstrating shear-thinning behavior. Incorporating UPs 1–3 solutions (0.2–0.6 % w/v) with rice flour increased gelatinization temperatures and modified pasting properties, increasing peak time, peak viscosity, and trough viscosity while reducing breakdown, final, and setback viscosities. Ulvan polysaccharide improved the viscous behavior of rice flour paste, indicated by increased loss modulus and tan δ (p > 0.05). Furthermore, ulvan polysaccharide improved the microstructure and texture of rice flour gel, with UP1 (0.6 % w/v) forming denser matrices and better texture. Molecular docking analysis suggested that hydrogen bonding is the primary interaction between rice glutelin and ulvan components.

Functional Diversification of Sec13 Isoforms for Storage Protein Trafficking in Rice Endosperm Cells.

Coat protein complex II (COPII) vesicles play crucial roles in mediating the endoplasmic reticulum (ER) exit of newly synthesized proteins to the Golgi in eukaryotic cells. However, the molecular functions of COPII components and their functional diversifications in plant seeds remain obscure. Here, we showed that the rice (Oryza sativa) glutelin precursor accumulation12 (gpa12) mutant is defective in storage protein export from the ER, resulting in the formation of aggregated protein bodies. Map-based cloning revealed that GPA12 encodes a COPII outer layer protein, Sec13a, that mainly localizes to endoplasmic reticulum exit sites (ERES) and partially localizes to the Golgi. Biochemical experiments verified that Sec13a physically interacts with Sec31 and Sec16, and mutation in Sec13 compromises its interaction with Sec31 and Sec16, thereby affecting the membrane association of the inner complex components Sar1b and Sec23c. Apart from Sec13a, the rice genome encodes two other Sec13 isoforms, Sec13b and Sec13c. Notably, we observed an abnormal accumulation of globular ER structures in the sec13bc double mutant but not in the single mutants, suggesting a functional redundancy of Sec13b and Sec13c in modulating ER morphology. Taken together, our results substantiated that Sec13a plays an important role in regulating storage protein export from the ER, while Sec13b and Sec13c are required for maintaining ER morphology in rice endosperm cells. Our findings provide insights into the functional diversification of COPII components in plants.

Enhancing mechanical and thermal properties of rice glutelin films through the interaction with pectin valorized from Akebia trifoliata var. australis waste

In this study, rice glutelin (RG) based films with improved mechanical and thermal properties were developed through the interaction of four different polysaccharides, pectin extracted by citric acid (CEP), so hydrochloric acid HEP, citrus peel pectin (CP), and λ-carrageenan (Car). Initially, the recovery of rice glutelin from rice and pectin from Akebia was performed. The results showed that hydrogen bonding, hydrophobic interaction, and electrostatic interaction may be involved in the interaction between RG and polysaccharides. At the same time, the addition of polysaccharides also improved the mechanical properties, thermal stability, and solubility of rice glutelin film. Among them, the mechanical properties of the RG-Car film were the best. The RG-HEP film showed the best thermal stability and the highest moisture content. RG-CEP film had a strong rigid structure, and the water content was significantly decreased compared with RG film. This study provided a theoretical basis for the development of protein-polysaccharide composite film.

The physicochemical properties and emulsion performance of different fractions from rice glutelin fibrils

Herein, oil in water emulsion was prepared by the different fractions from rice glutelin fibrils after centrifugal filtration. The morphology and physicochemical properties of retentates (RGFs), filtrates (FGFs), and total heated samples of amyloid fibrils from rice glutelin (TGFs) were characterized. As the oil fraction increased, the viscosity of TGFs-, RGFs- and FGFs-prepared emulsions increased, and the emulsions at φ = 0.7 exhibited the best stability. Compared to others, the RGFs stabilized emulsion showed a self-standing behavior, highest modulus (G′ of 1185 Pa and G″ of 211 Pa) and viscosity (6873 Pa s), due to the formation of dense interfaced layer. However, the hydrophilic FGFs particles cannot bind strongly to the interface after adsorption, resulting in a lower physical stability of emulsion. The TGFs-stabilized emulsion exhibited better centrifugation stability than other samples. These findings will provide further information into the tunable applications of different fractions from rice glutelin fibrils in food systems.

Development of pea protein nanoparticle/hydrolyzed rice glutelin fibril emulsion gels for encapsulation of curcumin

The potential of using emulsion gels stabilized by binary plant protein nanoparticle mixtures for the encapsulation and delivery of lipophilic nutraceuticals was evaluated. The particle characteristics, physical stability, water diffusivity, microrheology, large amplitude oscillating shear (LAOS) properties, and in vitro digestion of emulsion gels prepared by different ratios of hydrolyzed rice glutelin fibrils (HRGFs) and pea protein nanoparticle (PNP) were characterized. The emulsion gel with P/H = 2:1 (0.84 μm) exhibited the best storage stability and freeze-thaw stability, as seen by the smaller oil droplet size (1.02 and 1.42 μm, respectively). Low-field pulsed NMR indicated that the majority of water in samples was highly mobile. All the samples were predominantly elastic-like materials. The P/H 2:1 emulsion gel had the lowest FI value (6.21 × 10−4 Hz), the highest MVI value (5.57 s/nm2), G′/ G″ values and enclosed area, showing that it had denser 3D network structures, higher stiffness values, and a high sensitivity to changes in strain. Additionally, P/H 2:1 emulsion gel had a relatively high lipid digestibility (96.1 %), curcumin bioaccessibility (58.9 %), and curcumin stability (94.2 %). This study showed that emulsion gels stabilized by binary protein nanoparticle mixtures (PNP/HRGF) have potential as edible delivery systems for lipophilic nutraceuticals.

Elucidating the mechanism of amylolytic enzyme activity inhibition by rice glutelin amyloid fibril aggregates

Elucidating the mechanism of amylolytic enzyme activity inhibition by rice glutelin amyloid fibril aggregates

Molecular Spectroscopy Analysis of Different pH-Cycle Self-Assembly Between Rice Glutelin and Egg White Protein Under Neutral Conditions

Molecular Spectroscopy Analysis of Different pH-Cycle Self-Assembly Between Rice Glutelin and Egg White Protein Under Neutral Conditions

Structural characterization and binding interaction of rice glutelin fibrils complexing with curcumin

The rice glutelin (RG), the separated retentate (RGFs) and filtrate (FGFs) fractions from total glutelin fibrils (TGFs) at pH 3.5 were used as carrier for curcumin in this test. The solubility and antioxidant activities of curcumin were improved after binding with protein and fibrils. Compared to other complexes, the RGFs-curcumin complex exhibited a highest curcumin solubility (48.05%) and a superior sustained release property, probably owing to the stable hydrogen bond between the surface groups of fibrils and hydroxyl groups of polyphenols. In addition, thermodynamic parameters revealed that the RG/TGFs/RGFs-curcumin complexes were stabilized by hydrogen bonds and van der Waals forces, whereas FGFs interacted with curcumin through specific electrostatic interaction. Besides, after interacting with curcumin, the fibrils gathered into coarsened and agglutinated fibrillar aggregates, relating to the increment of a-helix and β-sheet structure. These results suggested that RGFs could be a good alternative for curcumin delivery in food industry.

Improving the functional properties of rice glutelin: impact of succinic anhydride modification

SummaryRice glutelin is poorly soluble which restricts its potential food application. Succinylation is a chemical modification that improves the solubility of proteins by derivatising the ɛ‐amino group of lysine residues. In this study, rice glutelin was modified by succinic anhydride at different succinic anhydride concentrations (10%, 20%, 30%, 40%, and 50%). The succinylated rice glutelin samples showed enhanced solubility, reduced particle size, and increased absolute zeta potential values. The emulsifying and foaming properties of succinylated rice glutelin were also significantly improved. Moreover, SEM revealed that the surface of succinylated rice glutelin had small pits and protrusions. These results suggest that succinylation can effectively improve the functional properties of rice glutelin, which will facilitate its application in the food industry.

Characterization of rice endosperm-derived antidepressant-like peptide (REAP): An orally active novel tridecapeptide derived from rice protein

It is ideal to ingest bioactive substances from daily foods to stay healthy. Rice is the staple food for almost half of the human population. We found that an orally administered enzymatic digest of rice endosperm protein exhibits antidepressant-like effects in the tail suspension test (TST) using mice. A comprehensive peptide analysis of the digest using liquid chromatography-tandem mass spectrometry was performed, and a tridecapeptide QQFLPEGQSQSQK, detected in the digest, was chemosynthesized. Oral administration of the tridecapeptide exhibited antidepressant-like effects at a low dose comparable to classical antidepressant in the TST. This also exhibited anti-depressant-like effect in the forced swim test. We named it rice endosperm-derived antidepressant-like peptide (REAP). Intriguingly, intraperitoneal administration had no effect. Orally administered REAP(8−13) but not REAP(1−7) exhibited antidepressant-like activity, suggesting that the C-terminal structure is important for the antidepressant-like effect. We confirmed the presence of REAP, corresponding to rice glutelin type B4(130−142) and B5(130−142), in the digest. The effects of REAP were blocked by both dopamine D1 and D2 antagonists. These results suggest that it exerts its antidepressant-like activity through activation of the dopamine system.Taken together, oral administration of a novel tridecapeptide exhibited antidepressant-like effects via the dopamine system. This is the first report of a rice-derived peptide that exhibits antidepressant-like effects.

Specific ions effect on aggregation behaviors and structural changes of amyloid fibrils from rice glutelin

Metal ions have been considered as an important factor on fibrils assembly. Herein, a comprehensive analysis of specific ions effect on fibril formation and structural changes was investigated. The addition of ions (except Zn2+) accelerated the aggregation kinetics of rice glutelin fibrils (RGFs) from 0.93 to 1.28–2.19 h−1. In addition, the fibrillization rate followed the order of NH4+ > Li+ > Na+ > K+ > Cu2+ > Mg2+ > Ca2+ > Zn2+. The highest yield and length of fibrils were observed with Ca2+, probably due to the ionic bridging effect and hydrated capacity of Ca2+. However, Cu2+ reduced the fibrils yield, which was attributable to the fact that Cu2+ disrupted β-sheet structure and inhibited the transition of monomer to fibrils. The polymorphism of fibrils was observed with different salts, and the light metals presented a superior effect on fibrils formation than heavy metals. Overall, this work will provide a further information into how to tune the structure of RGFs using various ions.

Production and characterization of basic fibroblast growth factor protein in rice suspension cultures

The basic fibroblast growth factor (bFGF) plays a major role in cell growth, survival, and other mitogenic activities. General applications of this protein involve tissue repair, morphogenesis, embryonic development, cellular growth, etc. Due to its importance and high demand, research has been done on producing recombinant bFGF in different expression platforms. We have produced bFGF in transgenic rice (Oryza sativa) cells growing in liquid suspension culture. The gene construct consists of the rice alpha-amylase (RAmy) 3D sugar-inducible promotor and rice glutelin secretion signal peptide for controlled production and release of the bFGF protein by manipulating the sugar concentration for batch or semi-continuous bioreactor runs. A thermal shift assay was performed on the purified rice cell produced bFGF (rbFGF) and the results were compared with E. coli derived bFGF. The purified rbFGF protein from rice cells was tested for propagating human-induced pluripotent stem cells (hiPSCs) up to eight passages. Furthermore, the biological activity measured in both crude lysate and purified rbFGF showed a similar pattern of the extracellular signal-regulated kinase (ERK) activity compared to E. coli derived bFGF (control), indicating that rbFGF is active before purification and the protein activity is not inhibited by other components of the lysate. From the current results, the rice-produced rbFGF protein is biologically active and could serve as a viable product for various applications.

Properties of Heat-Assisted pH Shifting and Compounded Chitosan from Insoluble Rice Peptide Precipitate and Its Application in the Curcumin-Loaded Pickering Emulsions.

Curcumin exhibits antioxidant and antitumor properties, but its poor chemical stability limits its application. Insoluble peptide precipitates formed by proteolysis of rice glutelin are usually discarded, resulting in resource waste. The coupled treatment of heat-assisted pH shifting and compounded chitosan (CS) was used to fabricate rice peptide aggregate-chitosan complexes (RPA-CS). The structure, interfacial behavior, emulsion properties, and digestibility of curcumin-loaded RPA-CS Pickering emulsions were investigated. Increasing the CS concentration led to lower interfacial tension but larger particle size, and the three-phase contact angle of the RPA-CS complexes approached 90°. Quartz crystal microbalance with dissipation (QCM-D) indicated that RPA-CS complexes with 6 g·kg-1 of CS (RPA-CS6) had the highest K1 (0.592 × 106 Hz-1) and K4 (0.487 × 106 Hz-1), suggesting that the softest interfacial layers were formed. The solid-liquid balance of RPA-RPA-CS emulsions was lower than 0.5, declaring that they had more elastic behavior than that of RPA emulsions. RPA-RPA-CS4-and RPA-CS6 emulsions had better storage stability, lower FFA release (79.8% and 76.3%, respectively), and higher curcumin bioaccessibility (65.2% and 68.2%, respectively) than RPA emulsions. This study showed that a low-value insoluble rice peptide precipitate could be used as a valuable emulsifier in foods, which may increase the economics and sustainability of the food supply.

Inhibition mechanism of rice glutelin on extruded starch digestion: From the structural properties of starch and enzyme activity

To increase the anti-digestion ability of extruded rice starch (ERS), the influence of rice glutelin (RG) on digestive and structural characteristics of ERS were investigated. The resistant starch content increased from 4.49 % to 18.08 % as the RG content increased, while the digestion rate and digestion velocity constant were reduced by the incorporation of RG. Morphological observations showed that ERS was adhered and encapsulated by RG, and the specific area of starch granules were decreased after the addition of RG. The results of XRD and FTIR suggested that the long-range and short-range orders of ERS were improved due to the complexation with RG. The thickness of crystalline of ERS was increased while its amorphous region thickness was reduced by the supplementation with RG. The 1H NMR and 13C NMR data revealed that the branching degree and double helix content of ERS was increased by 46.24 % and 52.67 % when RG content reached to 12 %. Additionally, the addition of RG altered the molecular weight and chain length distribution of ERS. The α-amylase activity and glucoamylase activity was inhibited by RG. These results could provide a valuable basis for the application of RG in extruded rice starchy foods with lower glycemic index.

Down-Regulation of Rice Glutelin by CRISPR-Cas9 Gene Editing Decreases Carbohydrate Content and Grain Weight and Modulates Synthesis of Seed Storage Proteins during Seed Maturation

The glutelins are a family of abundant plant proteins comprised of four glutelin subfamilies (GluA, GluB, GluC, and GluD) encoded by 15 genes. In this study, expression of subsets of rice glutelins were suppressed using CRISPR-Cas9 gene-editing technology to generate three transgenic rice variant lines, GluA1, GluB2, and GluC1. Suppression of the targeted glutelin genes was confirmed by SDS-PAGE, Western blot, and q-RT-PCR. Transgenic rice variants GluA1, GluB2, and GluC1 showed reduced amylose and starch content, increased prolamine content, reduced grain weight, and irregularly shaped protein aggregates/protein bodies in mature seeds. Targeted transcriptional profiling of immature seeds was performed with a focus on genes associated with grain quality, starch content, and grain weight, and the results were analyzed using the Pearson correlation test (requiring correlation coefficient absolute value ≥ 0.7 for significance). Significantly up- or down-regulated genes were associated with gene ontology (GO) and KEGG pathway functional annotations related to RNA processing (spliceosomal RNAs, group II catalytic introns, small nucleolar RNAs, microRNAs), as well as protein translation (transfer RNA, ribosomal RNA and other ribosome and translation factors). These results suggest that rice glutelin genes may interact during seed development with genes that regulate synthesis of starch and seed storage proteins and modulate their expression via post-transcriptional and translational mechanisms.

Multiple regulators were involved in glutelin synthesis and subunit accumulation in response to temperature and nitrogen during rice grain-filling stage

Rice glutelin is sensitive to temperature and nitrogen, however, the regulatory mechanism of glutelin response to temperature and nitrogen is unclear. In this study, we conducted the open field warming experiment by the Free-air temperature enhancement facility and application of nitrogen during grain filling. In three-year field warming experiments, glutelin relative content was significantly increased under elevated temperature and application of nitrogen. Temperature and nitrogen and their interaction increased the glutelin accumulation rate in the early and middle grain filling stages (10-25d after flowering), but decreased the glutelin accumulation rate in the middle and late grain filling stages (25-45d after flowering). Elevated temperature promoted pro-glutelin levels whereas application of nitrogen under warming increased the amount of α-glutelin. At the transcriptional level, the expression levels of the glutelin-encoding genes and protein disulphide isomerase-like enzyme (PDIL1-1), glutelin precursor accumulation 4 (GPA4), glutelin precursor mutant 6 (GPA2), glutelin precursor accumulation 3 (GPA3) and vacuolar processing enzyme (OsVPE1) of glutelin folding, transport and accumulation-related genes were up-regulated by nitrogen under natural temperature as early as 5d after flowering. However, elevated temperature up-regulated glutelin-encoding genes before 20d after flowering, and the expression of endoplasmic reticulum chaperone (OsBip1), OsPDIL1-1, small GTPase gene (GPA1), GPA2-GPA4 and OsVPE1 were significantly increased post 20d after flowering under warming. In addition, the increase in glutelin content worsened grain quality, particularly chalkiness and eating quality. Overall, the results were helpful to understand glutelin accumulation and provide a theoretical basis for further study the relationship between rice quality and glutelin under global warming.

Improved curcumin bioaccessibility in Pickering emulsion fabricated by rice glutelin fibrils

Pickering emulsion fabricated by food grade-solid colloidal particles as templates for delivering sensitive nutrients has attracted increasing interest in recent years. In this work, rice glutelin fibrils-stabilized Pickering emulsion was successfully fabricated for loading curcumin. In addition, the amyloid and non-amyloid structures were separated from the heated rice glutelin (RG) samples, and the emulsifying and encapsulating performances of these fractions were systematically observed. It was noted that the retentates of glutelin fibrils (RGFs)-stabilized Pickering emulsion exhibited a self-standing behavior, along with the highest curcumin encapsulation efficiency (94.17%) among other samples. Compared to the total heated glutelin fibrils (TGFs), a stronger gel strength was observed in emulsion containing purified fibrils, probably due to the closely arranged polyhedral emulsion structure. However, the filtrates of glutelin fibrils (FGFs) particles-prepared emulsion presented a weak gel strength due to the hydrophilic properties, accompanying by a surprisingly rapid release rate of curcumin during digestion. Nevertheless, TGFs-stabilized emulsion achieved a high curcumin bioavailability (35.06%) and sustained controlled release behavior. These findings proved that the sustainable and biocompatible protein amyloid exhibited excellent performance in encapsulating curcumin and improving curcumin bioavailability.

Hydrolyzed rice glutelin as plant-based emulsifier to reduce tween in emulsion facing process stresses and its application in coconut milk

The demand for natural plant–based emulsifier to reduce synthetic emulsifier is attractive to the food industry. The low emulsifying property of native rice glutelin (RG) can be improved by trypsin hydrolysis due to the partial unfolding of intact protein. The solubility and surface hydrophobicity of hydrolyzed RG at degree of hydrolysis (DH) 2.0 (HRG2) (89.48%, 7461.97) were much higher than RG (9.85%, 249.50). HRG2 was substituted for Tween 60 (Tw) in emulsion at ratio 100 : 0, 75 : 25, 50 : 50, 25 : 75, and 0 : 100. The selected 75 : 25 ratio emulsion was subjected to environmental stresses (pH, ionic strength, and temperature). The interfacial pressure of HRG2 and HRG2–Tw was in an increasing trend even after 3 h indicated the presence of HRG2 in the adsorbed interface. After amplitude sweep, the interface formed by HRG2 showed higher elastic modulus (Ed’) indicated a stiffer interface than HRG2–Tw. HRG2–Tw emulsion showed smaller mean droplet size with less concentration dependence than HRG2– and Tw–emulsion. HRG2–Tw–emulsion was precipitated at pI but was stable at pH > 6 and was stabilized under ionic strength similarly to Tw–emulsion. The pasteurized coconut milk with Tw (1%) had the lowest creaming index, followed by HRG2 (1%) and HRG2–Tw (1%) after 7 days which indicated the possibility to replace or reduce the use of synthetic surfactant in coconut milk.

Self-assembly mechanism of rice glutelin amyloid fibril aggregates obtained through experimental and molecular dynamics simulation analysis

In this study, intermolecular interactions and the self-assembly mechanism of rice glutelin amyloid fibril aggregates (RAFA) were studied using a combination of experimental and computer simulation methods. Based on the results of Thioflavin T fluorescence intensity, particle size measurement, and atomic force microscopy, the addition of NaCl and SDS accelerated the agglomeration of the RAFA, whereas adding urea caused the degradation of fibril clusters and the depolymerization of amyloid fibrils. The formation of the common cross β-sheet structure in the RAFA was primarily driven by hydrogen bonding, hydrophobic and electrostatic interaction were also involved. Visual molecular dynamics simulations indicated that randomly distributed peptides, hydrolyzed from rice glutelin, experienced aggregation, dispersion, structural rearrangements, and positional modifications to form the RAFA, which was compatible with the nucleation-elongation mechanism. The study supports for clarifying the self-assembly pattern of amyloid fibril aggregates derived from food protein, which is crucial for the denovo design and practical application of protein-amyloid fibril aggregates.